Poster: Diseases of Plants: Disease Detection & Diagnosis
Development of a loop-mediated isothermal amplification assay for the detection of Harpophora maydis
N. GONZALEZ (1), G. Munkvold (1) (1) Iowa State University, U.S.A.
Harpophora maydis, a seedborne fungal plant pathogen that causes late wilt of maize, is an important pathogen that occurs in Africa, Europe and Asia, but not in the Americas. It is a high-priority quarantine pathogen listed on the APHIS-PPQ OPIS pest list. Thus, there is a need for effective pathogen detection assays that can be used to identify contaminated seeds. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect H. maydis based on a specific AFLP sequence from GenBank. H. maydis isolates were grown on PDA and DNA was extracted with the Extract-N-Amp plant kit. A primer set (AFLP 17) consisting of 5 primers recognizing different regions of the target sequence was designed using Primer Explorer V4 software. The LAMP reactions were performed in a 25 µl reaction mix (1X Isothermal master mix) and incubated at 67°C for 25 minutes. The amplified products were detected by fluorescence and results were confirmed by gel electrophoresis. The LAMP assay showed specificity to the pathogen as shown by negative amplification when tested with other fungal species and was developed without the need to standardize DNA concentrations. The assay was effective for detecting the pathogen in seed samples containing inoculated seeds. This LAMP assay should be suitable as a fast, easy to perform and reliable method to detect H. maydis, providing a tool to help prevent the introduction of this potentially devastating pathogen into the USA.